Pseudomonas aeruginosa (PA) is a Gram-negative bacterial pathogen that infects a wide variety of host animals including humans. PA is a significant public health concern as a leading cause of hospital-acquired infections, burn-wound infections, and cystic fibrosis pathology. PA exhibits high tolerance for many commonly- used antibiotics, raising a clear need for a better understanding of PA virulence regulation. Historically, the field has focused on PA's response to chemical cues such as nutrients or signaling molecules. However, we have recently demonstrated that PA also regulates virulence in response to mechanical cues in its environment and that the ability to respond to mechanical forces may underlie PA's ability to target such a broad range of hosts. Here we propose to develop new technology to answer the key outstanding question: what is the physical mechanism by which PA senses mechanical force? We recently discovered that the retraction of type IV pili induce the expression of virulence factors through the Chp chemotaxis-like system. However, there is nothing known about how mechanical forces from retraction are transmitted to the Chp system. Therefore, the studies proposed here are intended to fill this void in our understanding of PA surface sensing. First, we will construct a combined optical-trapping, fluorescence microscope capable of applying controlled forces to single PA pili in live cells over long periods of time and monitoring the expression of key virulence factors. Second, we will use this system to define the effect of force on virulence factor expression in a variety of force ranges. Last, we will investigate whether motor driven retraction or tension within the pilus provides the signal that is read-out by the Chp system. The information gained by these studies will substantially further our understanding of PA as an opportunistic pathogen and how it senses solid surfaces as a way of identifying hosts. This knowledge will pioneer new interventions for treating this important pathogen.